Natural Killer T (iNKT) cells have evolved to recognize glycolipid antigens presented by CD1d molecules. Following stimulation through their T cell receptor (TCR), iNKT cells respond very rapidly, as is characteristic of innate rather than adaptive responses. iNKT cells have been implicated in the regulation of immune responses associated with a broad range of diseases, including autoimmunity, allergy, infectious diseases and cancer. iNKT cells can be activated through two major pathways. Microbe-specific CD1d-restricted lipids can stimulate iNKT cells directly. Alternatively, iNKT cells can be activated via recognition of CD1d-restricted self-antigens, in combination with inflammatory cytokines. This suggests that iNKT cells might play a role in the response to nearly all infectious agents. Furthermore, recognition of self by iNKT cells might play an important role in cancer immunity as iNKT cells have been shown to play a critical role in the immune surveillance of carcinoma, sarcoma and hematopoietic malignancies. Similarly, the inflammatory cascade triggered during ischemia-reperfusion injury and sickle cell disease appears to be a direct consequence of iNKT cells activation by self-derived lipids. Importantly, self-antigens that can trigger an iNKT cell response may be responsible for the positive selection of these cells during their development. The nature of the self-lipid(s) that are involved in these processes is currently a subject of controversy. We have engineered iNKT T cell receptors that conserve the same specificity than regular iNKT TCR but have a higher affinity for the antigen-CD1d complex. Using these unique tools, we have started to identify self-antigens that can bind the TCR when presented by CD1d molecules. Through distinctive and multi-pronged approaches we propose to examine what is/are the self-ligands of iNKT cells and how they are recognized. Finally, we will assess, using a highly innovative approach, how these newly identified self-ligands affect iNKT cell development. These studies will define the guidelines to optimize iNKT cell ligands and ultimately regulate iNKT cell function, with important implications for glycolipid-based vaccine development.